Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
  • C12N11/087—Acrylic polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/098—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer formed in the presence of the enzymes or microbial cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/815—Carrier is a synthetic polymer
  • Y10S530/817—Entrapped within the carrier, e.g. gel, hollow fibre

Definitions

• the present invention relates to a process for the preparation of an insoluble biocatalyst by reacting an amine with a compound possessing two or more acrylamide or methacrylamide groups, in the presence of an enzyme or an enzyme-containing cell substance.
• biocatalysts for carrying out enzymatic reactions, it is desirable in many cases to immobilize them beforehand.
• the biocatalyst is thus converted to an insoluble form.
• in an insoluble, finely divided form it can advantageously be used for a continuous reaction, for example in a column.
• the immobilized biocatalyst has the advantage that it can be reused because it can be readily separated off from the reaction solution, for example by filtration or decanting.
• immobilization increases the service life of the biocatalyst so substantially that only then does it become possible to use it economically.
• the biocatalyst forms a more or less physical bond to a carrier.
• the enzyme or the cell can be bonded to the carrier by ionic or adsorptive forces, or can be enclosed in a gel matrix or a membrane. In all these cases, it must be accepted that this type of bonding or enclosure is not permanent, and that the biocatalyst is gradually washed out during use.
• the biocatalyst is covalently bonded to the carrier.
• the biocatalyst generally in the form of a solution, is then brought into contact with a reactive carrier of this type, and covalent bonds are formed.
• the disadvantage in this case is that the capacity of the carrier is determined by the degree of functionalization, and is in general very low.
• the pore size has to be carefully matched with the biocatalyst to be immobilized.
• the shelf life of the functionalized carrier is in general limited.
• Another possible method of producing covalently linked insoluble (immobilized) biocatalysts consists in the preparation of a carrier in the presence of the biocatalyst. This can be carried out as follows: the biocatalyst is first functionalized with a suitable reagent and is then used as a reactant in the preparation of the carrier and incorporated into the latter by covalent bonds. The procedure is simpler if the biocatalyst does not need to be functionalized beforehand, but is incorporated by means of its own functional groups, for example amino groups, this incorporation into the carrier taking place during the preparation of the latter. For example, British Pat. Nos.
• 1,517,813, 1,518,746 and 1,541,100 describe the immobilization of biological material in polyurethane foams, where the reactive polyisocyanate reacts with the biological material on the one hand and builds up the foam on the other. Because of the high reactivity of the isocyanate prepolymer, it is appropriate in this case to pretreat the dry enzyme with this prepolymer; this greatly restricts the usefulness of the process.
• U.S. Pat. No. 4,334,027 describes the immobilization of cells or cell fragments in mixtures of epoxides and suitable hardeners. In this case, however, the process is greatly complicated by the use of an inert auxiliary polymer which influences the shaping of the catalyst and is subsequently removed. Moreover, the epoxides used are water-insoluble, and hence the usefulness of the system is in any case restricted.
• U.S. Pat. No. 4,288,552 discloses the preparation of immobilized biocatalysts by reacting the biocatalyst with glutaradialdehyde in the presence of a polyamine in aqueous solution, wherein the biocatalyst is incorporated into the network formed from glutaradialdehyde and the polyamine.
• the disadvantage in this case is the fact that the mechanical properties of the resulting network are unsatisfactory.
• Another restriction results from the fact that the polyamine must possess a high percentage of primary amino groups since only these, and not secondary amino groups, effect stable crosslinking with glutarodialdehyde.
• the present invention furthermore relates to a process for the preparation of an organic compound using an isoluble biocatalyst for the heterogeneous enzyme catalysis, wherein the insoluble biocatalyst is prepared as explained above.
• Suitable biologically active substances are enzymes or cells, and the latter may be intact or denatured. Disintegrated, digested or homogenized cells or cell fragments may also be employed. In the case of enzymes, either crude preparations or pure enzymes can be used.
• Preferred enzymes for the novel process are invertase, glucose isomerase, amyloglucosidase and alpha- and beta-amylase.
• Other suitable enzymes are oxidoreductases, e.g.
• alcohol dehydrogenase lactate dehydrogenase, aminoacid oxidase, peroxidase, catalase, glucose oxidase, alcohol oxidase, succinate dehydrogenase, glutamate dehydrogenase, uricase, phenol oxidase, catechol oxidase, monoamino oxidase, lipoxygenase, luciferase, nitrate reductase, nitrite reductase, chloroperoxidase, acetaldehyde dehydrogenase, aldehyde oxygenase, diaphorase, cholesterol oxidase, glutarthio reductase, hydroxysteroid dehydrogenase, xanthine oxidase, dopamine hydroxylase, cytochrome oxidase, diacetyl reductase, superoxide dismutase and limonate dehydrogenas
• polynucleotide phosphorylase dextran sucrase, phosphorylase, carbamate kinase, aminotransferase, transaldolase, methyl transferase, pyruvate kinase, carbamyl transferase, phosphofructokinase and dextran synthetase; hydrolases, e.g.
• a firm bond between the biologically active substance and the carrier is a bond, preferably a covalent bond, such that the biologically active substance is not washed out from the carrier, even over a long period (except, in certain cases, for a certain initial loss of biologically active substance which was only physically bonded).
• An insoluble biocatalyst is one which is suitable for heterogeneous catalysis, i.e. one which, because of its insolubility in the reaction medium (which as a rule is aqueous), its particle size and its sufficiently compact nature, can be readily filtered or separated off from the reaction medium in another manner. Cells and cell fragments are not included here because, although they too are insoluble, they are relatively difficult to filter.
• the concentration is not critical and may vary within wide limits.
• the concentration of the solution or suspension obtained is left unchanged.
• suitable water-soluble compounds possessing 2 or more methacrylamide or, preferably, acrylamide groups are N,N'-methylenebisacrylamide, N,N'-ethylenebisacrylamide and N,N'-diacryloylpiperazine as well as the corresponding methacryl compounds.
• the mechanical strength of the resulting gel can be improved if compounds or mixtures of compounds possessing (on average) more than 2, preferably as many as 6, reactive double bonds are employed.
• Such compounds can be obtained, for example, by reacting a bisacrylamide of the above type, inaqueous solution, with less than an equimolar amount of ammonia or one or more water-soluble amines possessing more than 2 hydrogen atoms bonded to the amine nitrogen.
• the molar ratio employed is such that there is more than 0.5, preferably from 0.7 to 1, mole of bisacrylamide per amine NH group.
• the resulting adducts also frequently possess improved water solubility, particularly if one or more of the amines used possess hydrophilic groups, e.g. ethanolamine.
• the water solubility of component B should be not less than 3, preferably not less than 10, % by weight at room temperature.
• Suitable water-soluble amines possessing 2 or more hydrogen atoms bonded to the amine nitrogen are ammonia, lower diamines, e.g. ethylenediamine, propylenediamine and tetramethylenediamine, and lower polyamines, e.g. diethylenetriomine and triethylenetetramine.
• component B possesses more than 2 acrylamide or methacrylamide groups, crosslinking also occurs when primary monoamines, e.g. ethanolamine, methylamine or ethylamine, or secondary diamines, e.g. N,N'-dimethylethylenediamine, ase used.
• the sum of the number of acrylamide or methacrylamide groups per molecule of component B and the number of NH groups per molecule of component C is more than 4, i.e. not less than about 4.1, advantageously 4.5 or 5, or preferably even higher.
• Very particularly preferred amino compounds are polymeric compounds, such as straight-chain or branched polyethyleneimine, or polyvinylamine. These amines substantially improve the mechanical stability of the immobilized biocatalyst.
• the immobilized biocatalyst is prepared by mixing the 3 components A, B and C.
• the two last-mentioned components are advantageously employed as aqueous solutions, the concentrations being preferably from 20 to 50% by weight.
• the molar ratio of the acrylamide or methacrylamide groups of component B to the amine hydrogen atoms of component C is preferably from 1:0.5 to 1:20.
• the biologically active substance (component A) is advantageously used as a solution or suspension but may furthermore be employed in the form of a powder.
• this substance is first mixed with a solution of component B, a certain time (from 1 minute to 2 hours) is allowed to elapse and a solution of component C is then added.
• a different sequence is also possible, depending on the particular case.
• Component C can be added in neutralized, partially neutralized or non-neutralized form, depending essentially on the stability of the pH of the biologically active substance. If the amino compound is employed in a non-neutralized form, the pH of the reaction mixture is in general very high (e.g. from 9 to 11). Under these conditions, gel formation is relatively rapid. However, such a high pH has a denaturing action on many enzymes, and it is therefore advisable to neutralize component C. A substantial advantage of the process described here is that gel formation occurs even at a relatively low pH, e.g. from 5 to 6, even if at a slower rate.
• gel formation can be carried out at elevated temperatures, e.g. 50° C., thereby accelerating it substantially. Temperatures below room temperature are of no advantage. Gel formation can take from one hour to 4 days, preferably from 2 to 8 hours. For economic reasons, longer times are not very useful.
• the gel can be comminuted by a conventional method, e.g. by passing it through a sieve, cutting it or extruding it. It can be dried, if appropriate freeze-dried, either before or after the comminuting process. Particle diameters of from 0.1 to 5 mm have proved advantageous.
• the mixture can be suspended in a stirred inert water-immiscible solvent, if appropriate with the aid of a conventional suspending agent.
• the mixture then forms solid beads, whose size can be varied within the above range (from 0.1 to 5 mm diameter) in a conventional manner by suitable choice of vessel, stirrer and stirring velocity.
• Particularly suitable water-immiscible solvents are aliphatic, cycloaliphatic and aromatic hydrocarbons, e.g. hexane, cyclohexane and toluene, and chlorohydrocarbons, e.g. 1,1,1-trichloroethane.
• suspending agents In order to effect better suspension, it is advantageous to add suspending agents to the aqueous phase. Suitable substances for this purpose are those used in reverse suspension polymerization, e.g. sorbitan esters. After solidification, the catalyst is filtered off and washed thoroughly with water or an aqueous buffer solution. Washing it beforehand for a short time with a water-soluble organic solvent, e.g. acetone or an alcohol, can be advantageous but is not necessary in every case.
• a water-soluble organic solvent e.g. acetone or an alcohol
• 25 mg of ⁇ -fructosidase were dissolved in 5 ml of 0.05 M sodium acetate solution at pH 5.3. To this stirred solution were added, in succession, 5 ml of a 20% strength solution of an adduct of 4,7,10-trioxatridecane-1,13-diamine with N,N'-methylenebisacrylamide (molar ratio 1:4) and 5 ml of a 25% strength aqueous solution of polyethyleneimine (MW 30,000; brought to pH 6.0 with hydrochloric acid). This mixture was left at room temperature for two days.
• the resulting solid gel was forced through a sieve to give particles of less than 500 ⁇ m size, and the product was washed with 0.05 M sodium acetate solution at pH 5.3.
• 2.5 g of the immobilized product (corresponding to 4.2 mg of the enzyme employed) were incubated for 30 minutes at 30° C. with 50 ml of a 30% strength sucrose solution. The conversion, determined polarimetrically, was 30%. This corresponds to a residual activity of 70 %, based on a corresponding amount of free enzyme.
• invertase activity 2.6 g of the catalyst (corresponding to 0.2 g of dry yeast) were shaken in 50 ml of a 30% strength sucrose solution (pH 5.3) for 90 minutes at 30° C. The conversion, determined polarimetrically, was 15%. This corresponds to a residual invertase activity of 50%.
• Amyloglucosidase was immobilized by a method similar to that described in Example 5. The yield of immobilized product was 38%.

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• Chemical & Material Sciences (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• Life Sciences & Earth Sciences (AREA)
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Citations (12)

• 1982
  • 1982-06-18 DE DE19823222912 patent/DE3222912A1/de not_active Withdrawn
• 1983
  • 1983-06-08 EP EP83105611A patent/EP0097281B1/de not_active Expired
  • 1983-06-08 DE DE8383105611T patent/DE3360089D1/de not_active Expired
  • 1983-06-15 JP JP58106014A patent/JPS596885A/ja active Pending
• 1987
  • 1987-04-28 US US07/045,759 patent/US4764467A/en not_active Expired - Fee Related

Patent Citations (14)

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